Pglo Lab
Genetic transformation of Escherichia coli with pGLO
(Adapted from: Biotechnology Explorer: Bacterial Transformation: The pGLO System. Instructors Guide. BIO-RAD).
Objectives a. To understand one of the most commonly used techniques for introducing DNA into E. coli cells and its use in molecular cloning. b. To become familiar with the concept of using green fluorescent protein (GFP) as a molecular tag for studying gene expression in bacteria and other organisms. c. To understand some of the mechanisms by which bacteria regulate gene expression.
Introduction
Genetic transformation is a process by which competent bacterial cells absorb DNA through their cell envelopes causing a change (i.e. a transformation) of their phenotypes. Some bacteria are naturally competent (able to take up external sources of DNA) and readily take up DNA (e.g. Streptococci), whereas others must be artificially induced to competence (e.g. Escherichia coli). The ability to artificially induce competence in E. coli, has become an invaluable technique for molecular geneticists who wish to genetically engineer DNA molecules.
In this exercise you will attempt to transform E. coli with a plasmid containing genes for phosphorescence, and for resistance to the antibiotic ampicillin. Plasmids are usually small, circular pieces of DNA that replicate independently of the bacterial chromosome. Many plasmids have been modified to function as vectors, or vehicles for transferring genes of interest from one organism to another. Plasmids that have been modified as cloning vectors usually possess a selectable marker gene such as an antibiotic resistance gene.
A typical DNA cloning experiment involves digesting the plasmid DNA of the vector with a particular restriction endonuclease that cuts it at only one position. Foreign DNA (e.g. human DNA) is then digested with the same restriction endonuclease. Because the plasmid and foreign DNA fragments were
(Adapted from: Biotechnology Explorer: Bacterial Transformation: The pGLO System. Instructors Guide. BIO-RAD).
Objectives a. To understand one of the most commonly used techniques for introducing DNA into E. coli cells and its use in molecular cloning. b. To become familiar with the concept of using green fluorescent protein (GFP) as a molecular tag for studying gene expression in bacteria and other organisms. c. To understand some of the mechanisms by which bacteria regulate gene expression.
Introduction
Genetic transformation is a process by which competent bacterial cells absorb DNA through their cell envelopes causing a change (i.e. a transformation) of their phenotypes. Some bacteria are naturally competent (able to take up external sources of DNA) and readily take up DNA (e.g. Streptococci), whereas others must be artificially induced to competence (e.g. Escherichia coli). The ability to artificially induce competence in E. coli, has become an invaluable technique for molecular geneticists who wish to genetically engineer DNA molecules.
In this exercise you will attempt to transform E. coli with a plasmid containing genes for phosphorescence, and for resistance to the antibiotic ampicillin. Plasmids are usually small, circular pieces of DNA that replicate independently of the bacterial chromosome. Many plasmids have been modified to function as vectors, or vehicles for transferring genes of interest from one organism to another. Plasmids that have been modified as cloning vectors usually possess a selectable marker gene such as an antibiotic resistance gene.
A typical DNA cloning experiment involves digesting the plasmid DNA of the vector with a particular restriction endonuclease that cuts it at only one position. Foreign DNA (e.g. human DNA) is then digested with the same restriction endonuclease. Because the plasmid and foreign DNA fragments were